Polymerase Chain Reaction (PCR), also known as in-vitro enzymatic gene amplification, is a nucleic acid amplification technology simulating natural DNA replication in vitro, has a very complex mechanism and can amplify target genes to a million times within a short period of time under the action of heat-resistant DNA polymerase and the guide of specific primers. The technology was invented by K. Mullis in 1983-1984 and has been widely applied in nucleic acid sequence analysis. After 30 years of development, PCR has been developed into a mature technology and has relatively small error rate in conventional experiments. However, in the actual applications, interference side reactions, such as base pair mismatch, amplification caused by formation of dimers between the primers and the like, often appear to a certain degree. These side reactions can cause non-specific amplification so as to result in low amplification efficiency when being slight, and cause amplification failure when being severe.
An important way for improving the specificity of the PCR amplification is to optimize the reaction components and the procedure of PCR. For many years in the past, many researchers made a lot of work in optimizing the reaction components of PCR, for example, the non-specific amplification problem can be avoided to a certain degree by adding DMSO, glycerine, betaine, nano-metals and the like into a reaction system. But in actual applications, some effects are not very ideal, for example, the excessive nano-metals and the like can inhibit the activity of the polymerase, thereby reducing the amplification efficiency.
By retrieval of existing patents and documents, it is found that the following contents are provided in Chinese patent application CN 101983241A: at least partially complementary modified (including thiol-modified and hydroxy-modified) oligonucleotide is subjected to strand hybridization with target nucleic acid, this way can be used for PCR amplification of the target nucleic acid, as it has been proved that more stable hydrogen bonding can occur between hydroxy nucleobases or thiol nucleobases and nucleobases in the target nucleic acid, the purposes of enhancing the modification sequence and the binding efficiency with a target sequence can be achieved. In the application mentioned in the patent application, the ‘at least partially complementary modified oligonucleotide’ is actually used as a primer in the PCR, the purpose of performing modification with hydroxy or thiol or other groups on the primer is to increase the binding efficiency of the primer with the target sequence and the primer does not have the function of enhancing the specificity of PCR.